Biology subjects

Brenna, S.

Publications and source records attributed to Brenna, S..

6 recordsLinked to original sources

bioRxiv2025

High-Purity Enrichment of Extracellular Vesicles from Diverse Sources by Conventional and Image-Based Fluorescence Activated Cell Sorters for Robust Downstream Applications

Selective enrichment of extracellular vesicle (EV) subpopulations from the heterogeneous EV pool is essential for understanding their characteristic biological functions and exploiting their potential as diagnostic and prognostic biomarkers. However, isolation of specific EV-subsets remains challenging. Fluorescence-Activated Cell Sorting (FACS) has emerged as promising technique for EV subpopulations enrichment, despite limitations associated to their small size. Although FACS-based EV sorting has been reported, a broadly applicable and systematically validated workflow is still lacking. Here, we describe and validate an optimized workflow for the sorting and analysis of EVs derived from diverse species, tissues, blood and cell culture systems. Using two advanced flow cytometric cell sorters, the BD FACSAria Fusion, and the BD FACSDiscover S8, we systematically evaluated key technical parameters, including nozzle size, sample dilutions, and sorting mode. The optimized workflow enabled efficient enrichment of differently labelled EV populations of interest, achieving near-100% purity, including rare subsets representing less than 10% of the total EV pool, while maintaining compatibility with downstream analyses. Sorted EV populations were characterized by high-sensitivity imaging flow cytometry, transmission electron microscopy, and liquid chromatography-tandem mass spectrometry. This workflow provides a robust framework for EV subset isolation and characterization, supporting both fundamental EV research and translational biomarker applications.

cell biology↗

bioRxiv2025

Binding of extracellular vesicles to stretched von Willebrand factor promotes platelet activation

Von Willebrand factor (vWF), promoting platelet aggregation in various diseases such as COVID-19, malaria and cancer, is a huge multimeric glycoprotein. This extraordinary size makes vWF a unique shear stress sensing molecule. Below a critical shear stress, vWF is in a globular conformation that prevents platelet binding. Above the critical shear stress, vWF is stretched into platelet accessible fibers. Although previous studies have suggested that leukocytes or cancer cells can bind to vWF fibers, acting forces and the likelihood of cell adhesion has remained largely unexplored. Here, we report that vWF is a size-selective protein that prefers to interact with objects smaller than 4 m in diameter. Consistently, tumor cell-derived extracellular vesicles (EVs) were able to interact with vWF in parallel to platelets. Although whole tumor cells under flow were unable to bind to vWF per se, binding of EVs and platelets along the vWF fiber promoted platelet aggregation, which in turn entrapped circulating tumor cells. In conclusion, our study highlights the shear-sensitive nature of vWF and its ability to bring EVs and platelets together to enhance coagulation. While EVs-vWF-platelet aggregates may serve as novel biomarkers, their therapeutic disruption may prevent hypercoagulation in disease.

cancer biology↗

bioRxiv2024

Efficient enzyme-free isolation of brain-derived extracellular vesicles

Extracellular vesicles (EVs) have gained significant attention as pathology mediators and potential diagnostic tools for neurodegenerative diseases. However, isolation of brain-derived EVs (BDEVs) from tissue remains challenging, often involving enzymatic digestion steps that may compromise the integrity of EV proteins and overall functionality. Here, we describe that collagenase digestion, commonly used for BDEV isolation, produces undesired protein cleavage of EV-associated proteins in brain tissue homogenates and cell-derived EVs. In order to avoid this effect, we studied the possibility of isolating BDEVs with a reduced amount of collagenase or without any protease. Characterization of the isolated BDEVs revealed their characteristic morphology and size distribution with both approaches. However, we revealed that even minor enzymatic digestion induces artificial proteolytic processing in key BDEV markers, such as Flotillin-1, CD81, and the cellular prion protein (PrPC), whereas avoiding enzymatic treatment completely preserves their integrity. We found no differences in mRNA and protein content between non-enzymatically and enzymatically isolated BDEVs, suggesting that we are purifying the same BDEV populations with both approaches. Intriguingly, the lack of Golgi marker GM130 signal, often referred to as contamination contamination-negative marker in EV preparations, seems to result from enzymatic digestion rather than from its actual absence in BDEV samples. Overall, we show that non-enzymatic isolation of EVs from brain tissue is possible and avoids artificial pruning of proteins while achieving a high BDEV yield and purity. This protocol will help to understand the functions of BDEV in a near-physiological setting, thus opening new research approaches.

neuroscience↗

bioRxiv2023

Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo

Synaptic loss is an early event in the undersupplied but not yet irreversibly injured penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. In the present study, we aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse the potential beneficial effect of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

neuroscience↗

bioRxiv2021

Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content related to inflammation and recovery processes

Extracellular vesicles (EVs) are lipid bilayer enclosed structures that not only represent a newly discovered means for cell-to-cell communication but may also serve as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 hours after experimental stroke in mice and compared them to controls (shams) using the nCounter(R) Nanostring panels, with or without prior RNA isolation from BDEVs. We found that both panels show similar results when comparing upregulated mRNA in stroke. Notably, the higher upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, indicating that regenerative mechanisms are already taking place at this time-point. The five top overexpressed mRNAs in stroke mice compared to shams were confirmed by RT-qPCR and, interestingly, were found to be present as full-length open-reading frame in BDEVs. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and probably predominantly present in small BDEVs ([≤] 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter(R) panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation and that the mRNA cargo of BDEVs indicates their participation in inflammatory but also recovery processes after stroke.

neuroscience↗

bioRxiv2019

BRAIN-DERIVED EXTRACELLULAR VESICLES ARE HIGHLY ENRICHED IN THE PRION PROTEIN AND ITS C1 FRAGMENT: RELEVANCE FOR CELLULAR UPTAKE AND IMPLICATIONS IN STROKE

Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischemic region. The fate of neurons in the surrounding penumbra depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24h after induction of transient ischemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs) whereas after ischemia, the main EV population originates from astrocytes. Moreover, sEVs presented high amounts of the prion protein (PrP) which were increased after stroke. Conspicuously, sEVs were particularly enriched in a truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or containing PrP (WT). We show that PrP-KO-EVs are rapidly taken up by neurons and colocalize with lysosomes. Although eventually WT-EVs are also found in lysosomes, the amount taken up by neurons is significantly higher for PrP-KO-EVs. Likewise, microglia and astrocytes were also engulfing PrP-KO-sEVs more efficiently than WT-sEVs. Our results provide information on the relative contribution of brain cell types to the sEV pool in mice and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to their cellular uptake.

neuroscience↗